The long term goals of this study are to definitively analyze the alterations in the in vivo pericellular matrix due to osteoarthritis and to determine how these changes affect cartilage maintenance and repair. Previous studies have reported that the pericellular region in osteoarthritic cartilage becomes enlarged and enriched in specific matrix molecules such as type VI collagen. These changes are important because all molecules which leave or enter the cell must pass through this interface between the chondrocyte and the cartilage matrix. During their passage, some of these molecules, such as growth factors, are modified or retained. The specific aims of this proposal are: (l) to determine how the molecular composition and organization of the pericellular matrix in osteoarthritic human cartilage differs from that in normal human cartilage, (2) to determine how the macromolecules enriched in the osteoarthritic pericellular matrix are associated with other matrix components and how these associations produce the observed matrix structure, (3) to relate changes in the in vivo pericellular matrix with alterations in chondrocyte metabolism in vitro, and (4) to determine how the structure - and composition of the pericellular matrix affects extracellular matrix assembly. The studies to accomplish these aims will make use of the PI's recently developed technique to conveniently isolate "chondrons": chondrocytes with their in vivo pericellular matrix and surrounding capsule. These chondrons are viable, obtained in good yield, and can be maintained in alginate head culture for several months. The in vivo pericellular matrix can now be studied independently of the surrounding interterritorial cartilage matrix. Chondrons and traditionally isolated chondrocytes will be obtained from normal and osteoarthritic adult human cartilage, maintained in alginate bead culture, and compared. The dynamic structure and composition of the pericellular matrix in living and fixed chondrons will be examined with confocal microscopy, nanovid microscopy (video-enhanced light microscopy combined with colloidal gold probes conjugated to monoclonal antibodies), and transmission electron microscopy. Chondrocyte metabolism in vitro will be studied with a variety of techniques including in situ hybridization, immunofluorescence, and SDS-PAGE with Western blotting. In addition to defining the changes in the pericellular matrix due to osteoarthritis, these studies provide for the characterization of an important new model system for studying the metabolism of osteoarthritic chondrocytes in vitro.